An 18-kDa hemagglutinin which possesses the property of inducing both aggregation of amebocytes and agglutination of erythrocytes has been isolated from limulus polyphemus amebocytes and purified by ion exchange chromatography. This non-glysolated, single chain polypeptide with an Mr of 18,506 and isoelectric point of 8.3 is stored exclusively in the large secretory granules of amebocytes. Based on the partial N-terminal amino acid sequence of 63 residues, DNA probes have been synthetized for screening a pBR 322 cDNA library constructed from limulus amebocytes. Northern blot analysis indicates the presence of a single mRNA. The primary structure derived from the cDNA sequence reveals an internal homology consisting of two consensus sequences, V-N/D-D/S-W-D and E-D-R-R-W. The formation of 5 disulfide bonds between ten 1/2 cys divides the molecule into three looped domains, each containing the E-D-R-R-W repeat. One of the novel features of this protein is that it shares 37 percent homology with a 22-kDa mammalian extracellular matrix protein isolated from fetal bovine skin. The two proteins exhibit a similar pattern of looped domains, each domain containing a homologous consensus sequence (i.e., E-D-R-R-W). The functional properties of the two proteins are similar in that the limulus 18 kDa protein agglutinates horse erythrocytes and aggregates limulus amebocytes, and the 22-kDa protein is an effective adhesion promoter for dermal fibroblasts.